III. Results
III.3. Cellular requirements, intracellular transport, and secretion of DHBV
III.3.3. Role of cholesterol and sphingolipids in DHBV morphogenesis
FDA Trypan
blue
control + Mevinolin
FDA Trypan
blue
control + Mevinolin
Fig. 76. Mevinolin has no cytotoxic effects on PDHs. Congenitally DHBV-infected PDHs were treated for 24 h with mevinolin. Subsequently, the viability test with trypan blue and FDA was performed.
Then, supernatants and cell lysates of mevinolin treated and non treated cells were analysed for viral protein-L and DNA. As shown in Figure 77, the secretion of DHBV in mevinolin treated cultures was comparable to that of the control cells and the intracellular levels of L protein and DNA were also similar. This indicates that a reduced cholesterol level does not interfere with the formation and secretion of progeny viruses. Unfortunately, the effect of mevinolin treatment on cellular cholesterol was not yet determined, nevertheless, under the experimental conditions used, the secreted viruses from mevinolin-treated cells were altered in their infectivity (Funk et al.,, manuscript in preparation).
These findings indicate that cholesterol is not required for viral formation, but is essential for the entry during de novo infection.
104 105 -_ + 48 h Mevinolin
Virions (5 x) Supernatants Celllysates L
rcDNA
p36
p28
rcDNA
1 2 3 4 5 104 105 -_ + 48 h Mevinolin
Virions (5 x) Supernatants Celllysates L
rcDNA
p36
p28
rcDNA
1 2 3 4 5
Fig. 77. Cholesterol depletion by mevinolin does not alter DHBV secretion. Congenitally DHBV-infected PDHs were treated for 24 h with 10 µM mevinolin. Supernatants and cells were harvested and subjected to immunoblot and PCR analysis for viral L protein and rcDNA, respectively.
Depletion of cholesterol from the PM is not critical for viral secretion
Lipid rafts are enriched on the surface of many cells, where they are exploited as platforms for virus assembly by influenza virus and HIV-1 (140-143). It has been shown that the transport of the influenza virus surface protein hemagglutinin (HA) from the Golgi to the PM was slowed in cells treated with methyl-β-cyclodextrine (MßCD) which depletes cholesterol (144). This indicates an essential role for lipid rafts in the transport and thus secretion of virus proteins. To test whether cholesterol and thus lipid rafts are involved in the transport of DHBV, congenitally DHBV-infected PDHs were treated with MßCD, which efficiently binds to cholesterol and extracts it from the cell surface (145). Cells were treated for 12 and 60 h with MßCD. The concentration used were previously shown to reduce the cholesterol level to 56,2% if treated for 1 h (146). Under these conditions, MßCD had no cytotoxic effect on the cells (Fig. 78).
FDA stain Trypan
blue
control MßCD
FDA stain Trypan
blue
control MßCD
Fig. 78. MßCD has no cytotoxic effects on PDHs. Congenitally DHBV-infected PDHs were treated for 12 h with MßCD and subsequently the viability test with trypan blue and FDA stain was performed
After 12 and 60 h of treatment with MßCD, supernatants and cells were harvested and analysed for viral L proteins and DNA.
_ +
rcDNA 12h MßCD
p36
p28 L
1 2
A
supernatants
Cell lysates Cell lysates
_ + 60 h MßCD
L
rcDNA
p36
p28
rcDNA actin
1 2
supernatants
_ + B
rcDNA 12h MßCD
p36
p28 L
1 2
A
supernatants
Cell lysates Cell lysates
_ + 60 h MßCD
L
rcDNA
p36
p28
rcDNA actin
1 2
supernatants
B
Cell lysates
_ + 60 h MßCD
L
rcDNA
p36
p28
rcDNA actin
1 2
supernatants
B
Fig. 79. MßCD does not interfere with secretion of DHBV. Congenitally DHBV-infected PDHs were treated for 12 and 60 h with MßCD. Supernatants and cells were harvested and analysed by immunoblots and PCR for of viral L proteins and rcDNA, respectively.
The secretion of DHBV was not altered by MßCD treatment as shown by both immunoblot and PCR analyses in figure 79A, while in figure 79B, a slight reduction of virions in the supernatant was observed. However, this was due to a reduced level of intracellular DNA in the treated cells in comparison to the control cells. One remarkable feature was that after MßCD treatment, the L protein pattern in the supernatants was changed. In the control cells, the hypophosphorylated L species is the abundant form of L (lower band), while only a minor band of hyperphosphorylated L was present (upper band). In the MßCD-treated cells, the hyperphosphorylated form becomes more abundant in comparison to the control cells. This indicated that MßCD treatment induces a hyperphosphorylation of L-protein in secreted viral particles. This could be the result of the activation of a specific kinase resulting in the hyperphosphorylation of L or to the inactivation of a phosphatase which is normally responsible for the dephosphorylation of L proteins.
It has previously been reported that L is specifically phosphorylated at serine 118 by ERK-type mitogen-activated protein kinases (MAP kinases) in response to extracellular stimuli such as exposure to low temperature, UV-irradiation, and mitogenic phorbolester (TPA) (52). To test whether MAP kinases were activated following MßCD treatment and thus responsible for the phosphorylation of L, the cell lysates from the above experiment were blotted for active MAP kinase as shown in figure 80.
active MAPK 1 2
3 4
Vimentin - + 12h MßCD
active MAPK 1 2
3 4
Vimentin - + 12h MßCD
Fig. 80. Hyperphosphorylation of L protein after MßCD treatment does not correlate with activation of MAP kinases. The same cell lysates as in figure 79 were blotted for active MAP kinase (MAPK) (upper panel) to test whether this might be responsible for the hyperphosphorylation of L following MßCD treatment. As a loading control, the same membrane was incubated with anti-vimentin (lower panel).
The immunoblot shown above indicates that the hyperphosphorylation of L proteins following MßCD does not correlate with activation of MAP kinases which are known to phosphorylate L proteins. Thus MAPK could be excluded as the responsible kinase for the observed effects. These results indicate that presumably other kinases are involved in L phosphorylation or that the observed effect was the result of the inactivation of phosphatases which normally dephosphorylate L proteins.
In conclusion, cholesterol depletion either with mevinolin or with MßCD has no effect on the assembly, budding, and secretion of progeny virus. However, cholesterol seems to be required for the infectivity of the virus.
Depletion of sphingolipids does not interfere with DHBV secretion
To further investigate the role of lipid rafts in DHBV morphogenesis, sphingolipids, essential components for the formation of lipid rafts, were depleted from PDHs using 2 independent chemical inhibitors Fumonisin B1 (FB1) and Myriocin (Myr). FB1 is a reported specific inhibitor of ceramide biosynthesis, a precursor lipid for all sphingolipids (147), while Myr inhibits the serine palmitoyltransferase, the key rate-limiting enzyme in sphingolipid biosynthesis (148). Treatment of PDHs for 3 days with 50 µM FB1 reduces the level of glucosylceramides and ceramids by 84 and 42%, respectively, while treatment with 10 µM Myr reduces these lipids by 40 and 11%, respectively (unpublished data, kindly provided by Dr. Funk, from the HPI, Hamburg and Dr. Brügger from the Biocenter Klein Flottbeck, Hamburg)
To test whether sphingolipids are required for DHBV morphogenesis, congenitally DHBV-infected PDHs were treated for 60 h with FB1 or Myr to assure an effective depletion of sphingolipids. Fresh medium containing the substances was added every 24 h to permit the secretion of viral particles that had been formed before the depletion had occurred. To show that these long period-treatments (60 h) were not toxic for the cells, a viability test was performed showing no differences between control and treated cells (Fig. 81).
Trypan blue
FDA
control FB1 Myr
Trypan blue
FDA
control FB1 Myr
Fig. 81. Sphingolipid depletion for 60 h is not toxic. Congenitally DHBV-infected PDHs were treated with FB1 and Myr for 60 h. To test whether these treatments are cytotoxic for the cells, a viability test was performed.
Next, supernatants and cell lysates were analysed by both immunoblot and PCR for the detection of viral L proteins and DNA, respectively. Secretion of DHBV was not altered by sphingolipid depletion and the intracellular levels of viral L protein and DNA was also comparable between treated cells and control ones. This indicates that sphingolipids depletion and thus lipid rafts are not involved in the morphogenesis of DHBV.
1 2 3 4 5 6 7 8 Unt FB1 Myr
103 104 105 106
-103 104 105 106 -L
rcDNA
p36 p28
rcDNA actin Extracellular
Intracellular
60 h treatment
1 2 3 4 5 6 7 8 Unt FB1 Myr
103 104 105 106
-103 104 105 106 -L
rcDNA
p36 p28
rcDNA actin Extracellular
Intracellular
60 h treatment
Fig. 82. Sphingolipid depletion does not alter DHBV secretion. Congenitally DHBV-infected PDHs were treated with FB1 and Myr for 60 h. Supernatants and cells were harvested and analysed for viral L protein and rcDNA.
Taken together, the results of this study indicate that cholesterol and sphingolipids are not required for DHBV morphogenesis and that lipid rafts seems to not be involved in the formation, transport, and exocytosis of DHBV. However, cholesterol is required for the infectivity of the virus.